Vasculogenesis is the de novo formation of blood vessels from mesoderm. Basic steps in the process of vasculogenesis are the generation of angioblasts from undifferentiated mesoderm and the coalescence and transformation of angioblasts into endothelial cells (ECs). In addition, our studies have revealed a subsequent step in the process that involves the fusion of small vessels to form large vessels and vascular sinuses. This process, termed vascular fusion, was discovered as a result of experimentation in which elevated levels of VEGF were shown to lead to uncontrolled fusion, hyperfusion. VEGF-induced hyperfusion has not only been described in embryos of multiple species, but importantly in adult neovascular processes. Indeed, uncontrolled fusion activity now stands as an impediment to the numerous therapeutic uses envisioned for VEGF. Understanding the mechanism(s) by which vascular fusion is controlled has been a focus of our research. As a result we have found that VEGF/VEGF receptor is critical to fusion and hyperfusion and that a correlative relationship exists between the density of ECs and specific vascular patterns. Based on these and other findings we have derived the hypothesis that regulation of EC numbers/density is fundamental to normal vascular fusion and the pathological process of hyperfusion. Major ways to influence EC numbers/density include control of mitosis, apoptosis and/or recruitment of EC progenitor cells from mesoderm. Experimentation outlined in this application will determine the morphological consequences of and the mechanisms by which VEGF-A (VEGF165/VEGFt21) and PLGF signaling via the VEGF receptors (Flkl, Fltl and Neuropilin 1 & 2) impact vascular morphogenesis and act to regulate EC numbers/density. With respect to recruitment of progenitor cells to sites of vasculogenesis, we will also extend on our preliminary findings indicating that circulating embryonic stem cells contribute to vasculogenesis and vascular fusion. A major strength of the proposed research plan is the use of both our well-established in vivo avian assay and a powerful new in vitro murine model of vasculogenesis that recapitulates salient aspects of in vivo vasculogenesis. The proposed research is expected to contribute to a greater understanding of vasculogenesis, vascular fusion, and hyperfusion and offer new perspectives for strategies that target the neovascular component of various diseases.